Oyi et al., Nig. Journ. Pharm. Sci., October, 2007, Vol. 6 No. 2, P.

14 – 20

Nigerian Journal of Pharmaceutical Sciences

Vol. 6, No. 2, October, 2007, ISSN: 0189-823X
All Rights Reserved

ANTIMICROBIAL SCREENING AND STABILITY STUDIES OF THE

CRUDE EXTRACT OF JATROPHA CURCAS LINN LATEX
(EUPHORBIACEAE)
* Oyi, A.R1. Onaolapo J.A1 Haruna A.K2. and Morah C.O1.

1Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences,

Ahmadu Bello University, Zaria – Nigeria.
2Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences,
Ahmadu Bello University, Zaria – Nigeria.

* Author for Correspondence: (pharmaroyi@yahoo.com)

ABSTRACT
A preliminary antimicrobial screening of the latex of Jatropha curcas was carried out. The stability of the liquid, dried
(powdered) latex and ethyl acetate extract (EAE) was also studied. The latex showed a broad spectrum of antimicrobial
activity against Bacillus subtilis. NCTC 8326, Escherichia coli NCTC 10418. Pseudomonas aeruginosa NCTC 6750.
Stapylococcus aureus NCTC 6571, Streptocococcus pyogenes NCTC 8198, Candida albicans. NCTC 3151A and
clinical isolates of Trichophyton sp., using agar and broth dilution methods. The diameter of zones of inhibition ranged
between 20 to 26 mm, when the activity of the latex was compared with that of established antibiotics, it was shown that
the latex seemed to be more active. The stability studies were carried out over 14 weeks under different storage
conditions. The I.R. spectra of all the samples were taken at two weekly intervals for four weeks to elucidate the effect
of light and moisture. It was also found that exposure to light decrease the antimicrobial activity with decrease in the
diameter of zone of inhibition. The decrease was as high as 10 mm over the period of 14 weeks of storage. The liquid
latex completely lost its activity over 48 hours. The pH value of latex and EAE also increased with time from 3.1 to 5.0
with accompanying decrease in aqueous solubility. The implications of these findings in the use of Jatropha curcas in
traditional medicine are discussed.

Key words – Latex, dermatophyte, preservative, photolabile, antimicrobial

INTRODUCTION However, one of the major problems of

Jatropha curcas Linn is commonly grown as
hedges and fences around gardens and
households in Northern Nigeria. It has been
documented to have medicinal uses for human
and veterinary purposes (Irvine, 1961). The
latex combined with the powdered leaves is
applied to sluggish wounds while when
formulated as enema it is used for the
treatment of gonorrhea (Irvine, 1961). It is
also used as an antiseptic against cuts and
wounds. The healing effect of curcain a
proteolytic enzyme from the latex on wound
has been demonstrated (Nath and Dutta,
1992).
natural products is instability. Stability of an
active substance is the capacity of that
substance in a specific container/closure
system to remain within its physical,
chemical, microbiological and toxicological
specification (Linter, 1975). Instability of
active ingredients could result from
interaction between active and inactive
components, enzymatic degradation,
environmental condition which include air (O2
and CO2), light, heat, water (hydrolysis) and
duration of time between storage and usage.
Degradation of products due to any of the
above could result in decrease in therapeutic

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Oyi et al., Nig. Journ. Pharm. Sci., October, 2007, Vol. 6 No. 2, P. 14 – 20
activity of product and appearance of toxic
substances as by-products of degradative
reaction.
An active product can be affected
chemically by radiation of a particular
wavelength only if it absorbs radiation of that
wavelength and the energy exceeds a
threshold. Ultraviolet radiation is said to be
the cause of many degradative reactions
(Linter, 1975).
An increase in temperature can also
cause an increase in the rate of chemical
reaction. This is true for all active products
and can lead to deterioration of some
thermolabile products.
In natural products, enzymatic degradation
also plays a significant role in the stability of
such products (Ferdinand, 1976). Changes in
the physico-chemical nature of active
molecules normally accompany degradative
changes e.g. pH, colour e.t.c
Jatropha curcas latex has been
reported to have strong antimicrobial activities
(Oyi et al. 2002), but is rather unstable leading
to loss of activity with time. So this work was
designed to determine the spectrum of
antimicrobial activity, compare with
established agents, look into the source of the
instability of the latex and proffer solutions to
it.
MATERIALS AND METHODS
Extraction
The liquid exudates from the cut stalk of
leaves and young stem were collected into
amber-coloured bottles from April to October
and stored at 40C until needed for use. To
obtain the powdered latex, it was spread in
thin layers over clean glass sheets and kept in
a dark cupboard to dry overnight (Irvine,
1961). The dried latex was subsequently
scrapped off the glass sheet with a sharp razor
blade. This was pulverized and packed in
amber coloured bottles.
A known quantity of the fresh latex (50 mls)
was successively extracted with 100mls each
of petroleum ether, chloroform, ethyl acetate,
methanol and water. The solvents of the
filtrates were distilled off with a rotary
evaporator.
Protection with Antioxidant and
Preservative
The freshly collected samples of latex were
protected separately with 0.1%w/v sodium
benzoate and 0.1%w/v sodium metabisluphite
and then stored at 40C. The antibacterial
activities of both were compared to that of the
unprotected latex stored at 40C and at room
temperature of 28oC to32o C.
Susceptibility Testing
Sterile nutrient agar plates were inoculated
with the diluted cultures of the test organisms
(103 cfu/ml) by flooding and excess discarded
(Anderson, 1970). The plates were dried for
10 mins in the incubator. Using No. 4 (8 mm)
sterile cork borer, holes were bored on the
plates and different concentrations of the latex
or extract (0.1 ml) for the liquid latex, 20 mg
for dried latex and Ethyl acetate extract (EAE)
were applied to different holes. One hour pre-
diffusion time was allowed after which the
plates were incubated at 37oC for 18-24 hours.
After the incubation, the diameter of zones of
inhibition was measured to the nearest
millimeter. For fungi incubation was at 250 C
for 5-7 days. For comparative purpose the
activities of some antibiotics were also tested.
The mean value of three experiments was
taken as the reading.
Determination of some physical changes of
the extract on storage
The pH of the aqueous solution of the latex
and EAE were taken at different time
intervals. A set of 10mg/ml solutions were
prepared and used for pH determinations. The
pH meter was zeroed using standard buffer
solution before analyzing the latex and the
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Oyi et al., Nig. Journ. Pharm. Sci., October, 2007, Vol. 6 No. 2, P. 14 – 20

extract solutions. To determine the solubility,
250 mg each of latex and EAE was added to 5
ml of distilled water in a 25 ml conical flask.
Dissolution was effected by turning the flask
continuously for 15 min. shaking was avoided
to prevent excessive foaming of the products.
The mixture was filtered through a pre-
weighed whatman No. I filter paper. The
residue and filter paper were dried at room
temperature (to constant weight). The
difference in weight between the filter paper
with residue and without residue was taken as
the weight of material that did not dissolve.
Infra-red (IR) Spectrophotometric Studies
The IR spectra of the samples of EAE were
taken for four weeks to elucidate the effects of
light and moisture.
The study was carried out at the
National Research Institute of Chemical
Technology (NARICT) Bassawa, Zaria.
RESULT
The latex has a broad spectrum of
antimicrobial activity, because it shows
inhibitory activity against Gram positive,
Gram negative bacteria and fungi (Table 1),
but the activity is higher on bacteria compared
to the fungi.

Table 1: Activity of fixed inhibitory concentrations of Latex of

Jatropha curcas Linn against 103 cells or spores/ml of Test organisms
Test organisms Zone of inhibition (mm)
Ps. aeruginosa 25
B. subtills 26
C. albicans 24
E. coli 24
Strept. pyogenes 25
Staph. aureus 21
Trichopyton sp. 20

A comparative study of the inhibitory activity antibiotics also showed a comparative activity
of the latex with some commonly prescribed (Table 2).

Table 2: Comparative Inhibitory activities of the fixed concentrations of Latex with

commonly prescribed antibiotics
Agents Zone of Inhibition (mm)
E. Ps. Strept B. substillis Staph. C. albicans Tricophyton sp.
coli aeruginosa pygenes aureus
Latex 24 25 25 26 21 24 20
Tetracycline 30ug 8 20 16 8 16 8 NT
Gentamicin 10ug 20 19 15 18 23 19 NT
Pen.G 10 I.U 8 10 8 8 16 13 NT
Procaine Pen 10 I.U NT 10 8 8 16 13 NT
Fluconazole10 mg NT NT NT NT NT NT 40
Griseofulvin 10 m NT NT NT NT NT NT 19
Note: Diameter of cork borer is 8 mm
All readings are mean of three experiments
NT – Not tested

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There was slight difference in the diameter of and unprotected latex (Table 3). These
zones of inhibition produced by the protected differences were found to be statistically
insignificant at 99.05% confidence level.

Table 3: Effect of protection with 0.1% w/v sodium metabisculphite and 0.1% w/v
sodium benzoate on the antibacterial activity of latex
Period of Organism with zone of inhibition (mm)
Storage (weeks)
E.coli Ps .aeruginosa Stept. pyogenes S.aureus B. subtilis
SM C SB SM C SB SM C SB SM C SB SM C SB
1 23 21 22 24 24 25 24 22 24 24 22 23 24 21 25
2 23 20 20 24 21 23 24 20 24 24 22 22 24 19 22
3 23 19 19 23 18 23 23 18 23 21 19 20 23 16 21
4 19 16 16 20 16 20 21 NI 20 21 18 20 21 15 18
5 17 14 14 19 13 19 20 NI 17 19 17 17 18 10 17
Control: 0.2ml of 0.1%w/v of sodium benzoate had no activity on all the organisms.
0.2ml of 0.1% w/v of sodium metabisulphite had no activity on all the organisms
Key:
a. SM = Latex with sodium metabisulphite (0.1% w/v)
b. SB = Latex with sodium benzoate (0.1% w/v)
c. C = Unpreserved Latex
d. NI = No Inhibition

There was general reduction in the activity of
latex and EAE with time, however, this
reduction is more pronounced within the first
two weeks of storage. The liquid latex lost all
its activity within a week of storage while the
dried latex retained its activity for up to 14
weeks period of study.
The aqueous solubilities of latex and EAE
were found to be affected on storage. The
solubilities decreased while the pH value
increased. Table 4 gives details of the loss of
activity with time under varying storage
conditions.

Table 4: Susceptibility of E. coli and Staph aureus to 30mg of the

latex and EAE under different storage conditions and time
Duration (weeks) Diameter of zone of Inhibition (mm)
E. coli Staph. Aureus
A1 A2 B1 B2 C1 C2 A1 A2 B1 B2 C1 C2
0 38 38 36 36 38 36 32 32 24 24 32 34
1 30 33 30 29 34 32 26 28 30 29 28 32
2 23 30 21 26 33 30 24 28 27 28 28 28
4 22 26 20 25 33 28 24 26 25 27 28 27
6 21 26 20 24 28 27 24 26 23 25 28 27
10 21 25 20 24 28 27 24 26 23 25 28 25
12 20 25 18 23 28 26 23 26 22 24 28 25
14 20 25 18 23 28 26 23 26 22 24 28 25
Key:
AI = EAE in colourless bottle ; A2 = EAE in coloured bottle
BI = Whole latex in colourless bottle; B2 = Whole latex in coloured bottle.
CI = Desiccated EAE in coloured bottle; C2 = Desiccated whole latex in coloured bottle

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However latex is more soluble than EAE have different pH values. With increase in
(Table 5). The latex dried at room temperature temperature, the pH values increased (Table
(28 – 32o C), 500 C and 1000 C were found to 6) and solubilities decreased.

Table 5: Solubilities and pH Values of Latex and EAE with Time

Substance and Period of Storage (weeks)
colour 14 weeks
pH Colour Solubility (mg/ml) pH Colour Solubility (mg/ml)
A1 4.1 off-white 22 5.1 brownish-red 11
A2 4.1 off-white 22 4.6 brownish-red 15
B1 3.1 off-white 30 4.9 brownish-red 20
B2 3.1 off-white 30 4.3 brownish-red 25
C1 4.1 off-white 22 4.4 brownish-red 18
C2 3.1 off-white 30 4.0 brownish-red 27
KEY:
A1 - EAE in colourless bottle
A2 - EAE in coloured bottle
B1 - Whole latex in colourless bottle
B2 - Whole latex in coloured bottle
C1 - Desiccated EAE in coloured bottle
C2 - Desiccated whole latex in coloured bottle

Table 6: Effect of temperature on pH and solubility of latex

o
Temperature ( C) pH Solubility (mg/ml
28 – 32 3.4 28
50 4.0 25
100 4.7 13

Table 7: IR peaks of samples AI, A2 and CI after two and four weeks
of storage
Sample Peak at 2 weeks (cm-1) Peak at 4 weeks (cm-1) Tentative assignments
A1 3282 3265 -C-H stretch
1461 1455 -C-H stretch
1377 1376 -C-H stretch
1174 1170 -C-H stretch
1612 -C=C stretch
A2 3323 3257 -O-H stretch
1455 1461 -C-H stretch
1167 1377 -C-H stretch
1612 -C=C stretch
C1 3323 3257 -O-H stretch
1459 1464 -C-H stretch
1158 1111 -C-H stretch
A1 - EAE in colourless bottle
A2 - EAE in coloured bottle
C1 - Desiccated EAE in coloured bottle

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DISCUSSION
The latex showed a good antimicrobial
activity against all the tested organisms. The
activity of the latex against both Gram
negative and Gram positive bacteria is
comparable; showing its broad spectrum of
activity. The latex also showed a very good
activity against Candida albicans and
Tricophyton sp. The antimicrobial activities of
the latex could be due to the presence of
secondary metabolites such as tannins,
flavonoids and saponins which have been
confirmed to be present (Levens et al., 1979).
Tannins coagulate the cell wall
proteins resulting in bactericidal activity in
high concentrations, while saponins are
surface – active agents; they alter the
permeability of the cell wall thus facilitating
the entry of toxic materials or leakages of vital
constituents from the cell. Flavonoids are
phenolic in nature, they act as cytoplasmic
poisons. They inhibit the activity of enzymes
(Iwu et al., 1990; Pathak et al., 1991). The
antibacterial activity of the latex seems to be
as a result of the combined effects of the
metabolites. The addition of sodium
metabisulphite and sodium benzoate was
intended to take care of oxidation and
microbial contamination while storage at low
temperature was to retard the rate of
deterioration. The activity of the latex reduced
with time in both protected and unprotected
cases (Table 3), a statistical analysis of the
data revealed that these differences in activity
were not significant at P<0.05%. indicating
that oxidation and heat may not be the only
degradative pathways involved in the
degradation of the latex, other mechanisms are
involved.
The liquid latex lost all its activity
within a week, while the dried latex
maintained its activity for up to fourteen
weeks period of study. This indicates that
hydrolysis plays a major role in the
degradation of the latex. The main target
classes of hydrolytic reactions are the amides,
esters and lactams (Florence and Attwood,
1981), suggesting that these compounds may
be the main components of the latex.
The dried latex lost its activity gradually on
exposure to light (Table 4) suggesting also
that the active components of the extract are
also susceptible to photochemical degradation.
The aqueous solubility of samples AI, A2 and
C2 were also found to be affected by
deterioration. The solubility decreased with
increase in pH values with the development of
colour changes (Table 4). The application of
heat to dry the latex also produced
physicochemical changes in the nature of the
latex (Table 6). Changes in the chemical
nature of substances are usually accompanied
by physical changes as evidenced by the
changes in pH, colour and solubility.
The antibacterial activity reduces with
time of storage (Table 4) confirming that the
initial compounds with acidic pH are more
active than those with high pH value (pH
changed from 3.1 to 5.1)
The IR spectrum of EAE indicated the
presence of aromatic phenolic compounds and
phenolic compounds are known to generally
behave as acids (Gisvold, 1977) with high
antimicrobial activity. The biological
activities of phenolic compounds are greatly
affected by pH. At low pH the compound
remains in the unionized form, which is more
lipid soluble than the ionized form and can
gain more entry through cell membranes.
Phenolics are susceptible to attack by oxygen
of the air and by oxidizing agents, which
converts them to the corresponding quinones
(Gisvold, 1977). The degradation of the latex
by hydrolysis and heat may suggest that the
main active components of the latex are
phenolic in nature, undergoing hydrolysis and
oxidation in the presence of unsuitable
conditions.
A close examination of the IR spectra
of the samples indicates that new peaks which
were not present in the initial samples
appeared on storage of the samples e.g new
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Oyi et al., Nig. Journ. Pharm. Sci., October, 2007, Vol. 6 No. 2, P. 14 – 20
peaks at Cm-1 1461 and 1377 were observed
after two weeks and at Cm-1 1455 and 1376
after four weeks of storage. While for sample
A2 new peak at Cm-1455 after two weeks and
Cm-1 1461 and 1377 after four weeks (Table
7). For sample C1 at 1459 and 1464 after 2
weeks and four weeks respectively. Although
spectral changes were observed in all the
samples, C1 retained the closest features to the
original EAE spectra. This suggests that all
the samples are undergoing degradative
changes but at different rates. Between four
and fourteen weeks of storage the active
compounds in the sample seem to have
stabilized. Therefore, giving more uniform
antibacterial activities (Table 4). Enzymatic
activities may also have been taking place in
the powdered sample as the latex has been
confirmed to contain some enzymes (Nath and
Dutta, 1992, Auvin et al., 1977). Enzymes are
often present in natural products and active
even in solid state and the presence of
moisture accelerates their activity. It can be
concluded that the latex and ethyl acetate
extract of Jatropha curcas are highly unstable
under ambient storage condition and that the
antimicrobial activity of these products
decrease with time. The instability of these
products is catalyzed or accelerated by factors
like moisture, temperature and light. For a
stable product to be obtained, these factors
must be excluded. Traditional healers using
this product must come to term with the fact
that this product is very unstable, particularly
those in aqueous solution and efforts must be
made to find solutions to the instability of this
product which inspite of its unstatic potency
still find a good use in traditional medicine.
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Ferdinand, W. (1976): The enzyme molecule. Printed in
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Florence, A. T. and Attwood, D. (1981). Chemical
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